RESOLVING POWER, MAGNIFICATION, AND EVOLUTION OF THE MICROSCOPE OBJECTIVE.

AUTHORS: Jurriaan de Groot and Barry Sobel, with the help of James Solliday.

EDITORS: Joe Zeligs and Barry Sobel

INTRODUCTION
From the earliest days of the microscope, microscopists yearned for higher resolution, i.e. the ability to see more detail. Erroneously, it was believed, that this could be achieved simply by means of higher magnification. Although producing higher power lenses is a matter of making lenses with smaller and smaller radii, as the magnification increases, so do optical aberrations. Furthermore, when lenses are combined, as in a compound microscope, or even within an objective, any aberrations will be multiplied.

Although the first microscopes were made in the 1590's, it was not until the 1870's that it was realized, that the perfect objective brings together in one focal point the light rays received from the object by the entire surface of the front lens, across the whole of the visible color spectrum. This, in order to ensure that no visual information is lost, which can contribute towards formation of the image. Any shortfall in achieving these goals is considered an aberration, and leads to a deterioration in the quality of the image, and hence, resolution. It is important to note, that this perfect objective has never been achieved, although modern plan-apochromats come very close. Before this paradigm shift, simple microscopes using single lenses of various types showed less distorted images than compound instruments at the same magnification, because the multiple lenses in compound microscopes multiplied the aberrations. This web page is an attempt to illustrate the various types of microscope objectives found with compound microscopes and review their evolution over time. We start our discussion in the 1700s, the period from which time onward, a number of examples are still extant.

OBJECTIVES IN THE 1700s

Early objectives for compound microscopes were often encased in ivory or even wood. The objective shown here to the left contains a single biconvex lens in blackened ivory mount. According to Dr Joe Zeligs, it probably dates from the 1730s and may have belonged to an early form of Culpeper microscope, perhaps by Mathew Loft or one of his conteporaries. The front aperture is smaller than the lens, and the rear is smaller still. This was a common method to reduce aberrations, albeit at the expense of resolution. This lens is marked with two dots and also the number 2 on the threaded end, indicating that it was the second to highest power of a set for the microscope it was made for. When viewed under a stereo microscope, this lens has some scratches and scuffs on both surfaces. This pre-achromatic objective produces fair images, but with expected chromatic and spherical aberration.

One or two decades later, pre-achromatic objectives were made of brass, whereby a biconvex or planoconvex lens was contained in a dish shaped cell and held down loosely by a screw cap, as in the objective from a numbered Benjamin Martin microscope shown on the left. The lenses were often obtained from the trade, and were ground by hand by people, sometimes children, working from home, so tolerances could be wide, and the surface often fraught by imperfections. Worse still, as the lens was loose (some rattle when handled), centering was poor, which all led to an increase in aberrations. These were not well understood, and many observers, using these objectives, believed they saw actual structures that were, in actuality, nothing more than severe distortions that did not represent the actual structures observed. Because of the high magnification, uncorrected for aberrations, many objects appeared to be nothing but vague round blobs. Some observers, such as Milne-Edwards went so far as to believe that all objects were composed of "globules". Today we refer to magnifications which show no more fine detail as empty magnification.

Objective lenses with an even higher magnification and very small radius, too small to be contained in brass cells, were clamped between 2 thin domed brass plates onto which a protective cap could be screwed. This method of fixation was also used in simple microscopes. An example of such an objective is shown here. As expected, when used on a compound microscope, these had a short working distance, and good examples resolve the striations on the scales of Lepisma Saccharina (required N.A. 0.10 - 0.13).

For the observation of opaque objects, pre-achromatic objectives were constructed featuring a silver mirror in which a central aperture, behind which the chromatic lens was mounted. In these, access to the lens itself is gained by unscrewing a small ring at the rear of the assembly. This type of objective was originally popularized by Johannes Nathaniel Lieberkuhn (1711-1756), a physician, who used this design first on a simple microscope incorporating a small silver speculum, which had to be of a similar focal length as the objective lens, in order to reflect the maximum amount of light on the specimen when in focus. The image to the left shows 2 Lieberkuhn objectives of different focal lengths from the set as also shown belonging to a George Adams microscope Ca 1790.

During the course of the pre-achromatic period, a number of chracteristic compound microscope types were developed, each employing somewhat different methods to affix the objective to the nose piece. In Culpeper and Cuff types, female objective threads were frequently used, whereas Benjamin Martin used male objective threads on his Universal models. George Adams, and his successors, W. and S. Jones, also used male threads, and their "Jones Most Improved" model continued to be made and retailed by other opticians into the 1830's. The objective shown on the left is from one of these. The ubiquitous "Martin type drum" microscopes, which continued to be sold into the 1870's also had this same objective thread configuration.

A completely different method of mounting multiple objective lenses was first used on solar and simple microscopes, but soon also in compound instruments. These "objective sliders" often featured 4 or 6 biconvex lenses clamped between two longitudinal brass plates, sliding in a dovetailed ring which was affixed to the arm of the microscope. A sprung pawl acted on notched recesses, to ensure that once chosen, each lens would be centered at the end of the optical tube. One such slider from a chest microscope retailed by Schmalcalder is shown to the left.

This era also saw the first use of a rotating nosepiece, in which a number of biconvex lenses were held between two circular brass plates instead of a slider. This feature first appeared on Adams's "New Universal double microscope" (left), but W. and S. Jones and then others, later employed a much more compact version on their "Most Improved" model, as seen in the examples shown to the right. Later on, as objectives became more complicated, heavier, and longer, nosepieces were made to accomodate objectives of the user's choosing, intiially as a nosepiece double objective changer, and later, in the 20th century, accomodating up to six objectives in a shallow cone-shaped device.

In terms of optical development, the use of more than one lens in objectives certainly precedes the achromatic era. Benjamin Martin used a between-lens placed at the top of the snout carrying the single lens objective, whereby this lens, which was often 4 or 5 inches in focus, functioned as the back lens for the whole series of objectives. This reduced the working distance of the objective, and effectively brought a slight improvement in its angle of aperture.

Anticipating the later use of "achromatic buttons", some makers resorted to the use of stacked biconvex non-achromatic lenses contained in brass cells, which screwed onto each other. This divided the amount of spherical aberration of one lens over several lenses with bigger radii, while also providing a selection of magnifications with the use of different lens combinations. The objective shown on the left is from a Dutch chest microscope C.1780, and has a front lens marked '2' and back lens marked '3' in engraved dots. The other objective, shown to the right, features 3 non-achromatic buttons, each holding a biconcave lens of the same focal length, belongs to a chest microscope retailed by Mathew Berge,successor to Jesse Ramsden,and can be dated 1800-1819. Astonishingly, objectives made up of multiple small non-achromatic lenses separated by little fiber rings continue to be made today, and can be found in cheap toy microscopes, often sold as kits for children. Inevitably, these produce much empty magnification, which does little to promote microscopy as a hobby.

It is not well known that even before the advent of achromatics, there was a period of experimentation aimed at improving the resolution of the pre-achromatic objective. To date, the authors have not been able to find a reference to the mystery objective shown on the left, which is made up out of 2 positive meniscus lenses*, the front element being of clear- and the back lens being made of blue-green glass. The concave surfaces are turned towards the object, which hints at an attempt to correct spherical aberration, while the colored glass may have been aimed at making it monochromatic, and hence less suceptible to chromatic aberration. Focal length is about 5 mm, and, being not stopped down, its N.A. is about 0.25. Only part of this aperture can be utilized, as the maximum resolution of about 2 um can only be achieved with a narrow cone of light. The outer diameter of the threads of this objective is 15.5 mm and the thread pitch is 36 t.p.i.

*Meniscus lenses are concave on one side and convex on the other. A positive meniscus lens is thicker in the center than at the edges.

The objective shown here achieves a similar performance via simpler means, and consists of a plano-convex lens contained in a flat brass ring, which is held in place by a screw-on external stop, which is placed before the rear lens, which has its flat surface towards the object. Again, the working distance is short, but due to the placement of the stop more light rays from the object can be utilized, than would have been the case, if such a small aperture had been located immediately behind the lens. Resolution, as measured using a Graticules 50x2 um slide, is also 2 um. It has the same thread diameter and pitch as the prior entry.

The third objective, which came with the 2 pictured in the previous 2 paragraphs, features a low-power plano-convex lens, but is different in shape than most objectives from the pre-achromatic era. All 3 objectives shown here carry the same 15.5 mm diameter thread, and it is tempting to speculate, that they were once used on the same microscope. The big question remains, as to whether they pre-date the appearance of the first achromatic doublet objectives, or were made around the same time by someone seeking to replicate their improved resolution via other means? This objective has the same thread diameter and pitch as the prior two entries.

Although, as discussed later, the work of C.R. Goring (1793 - 1840) on the improvement of the achromatic objective, and the use of test objects has received attention in a number of publications, his earlier experiments aimed at improving the image of the pre-achromatic compound microscope has all but slipped into obscurity. Having originated from the south of London, he attended Edinburgh University from 1812, graduating as a Doctor of Medicine in 1816. There, he may well have met David Brewster, the reknowned polymath, and author of A treatise on new Philosophical Instruments, which was published in 1813, and contained a section on experiments with chromatic microscope objectives used in an immersive fluid, as to reduce their chromatic aberrations.

In 1819 Goring published his own paper Description of an improved Microscope (Thomson Annal of Philosophy; Vol. 13, pp. 52-59), describing his version of a microscope(left), which can be used for the examination of opaque, and transparent objects, and which, in inverted form, can be used as a solar microscope, projecting its image inside a box acting as a camera obscura. Its objective consists of two biconvex lenses, mounted at a certain distance, having an aperture stop in between. It is explained that the latter configuration is similar to those used for the eye-pieces of refracting telescopes (the image inverting part). In this earlier version, three different astronomic eye-pieces are used to increase the magnification. Goring states, that this instrument was made by that acute and distinguished artist, Mr. Adie, of Edinburgh,.., and features a large oval mirror, as well as an under stage condensing lens. The objective is obviously stopped down to a lesser extent, than was usual at the time, as Goring asserts that it provides an abundance of light when viewing opaque objects.

It appears that Goring continued to experiment with chromatic objectives over successive years, as in 1824 he publishes another paper (Quart. J. of Science, Litt. and the arts, Vol. 17 pp. 202-209) where he describes a set of improved microscope objectives(right) with focal lengths ranging from 2 to 1/2 inch, which consist of 2 plano-convex lenses, with their convex surfaces turned inwards, and whereby a stop is placed in the focal plane of the front lens, i.e. where the rays cross each other. The back lens (with a focal length between 3 to 2 up to 2 to 1 times the focal length of the front lens) is placed closely above the stop. Goring claims that: these object-glasses I can confidently recommend as greatly superior to those in common use; they are bright, clear, and distinct, free from spherical aberration, and will shew no sensible colour with opaque objects of any kind.., but: When, however, they are made to view an object illuminated from behind, which does not suffer the light to pass through it while its edges are seen, as for example the legs of some insects, some kinds of moss, &c, which have little transparency, the uncorrected colour is then decidedly seen. Goring reports, that this type of objective only works well up to focal lengths of up to 1/4 inch, and had a number of silver cups made for holding very deep single lenses (of shorter focal length) intended to view opaque objects, which, together with the object-glasses before-mentioned, were executed for me by Mr. Tuther, optician, in High Holborn.. (see illustration, right). By now, Goring had obviosly moved back to London, as he also states that: Mr. Varley and Mr. William Tulley, of Islington, are the only individuals who can make such deep lenses as they ought to be made. Around this time, he also meets Andrew Pritchard, with whom, for awhile, he worked on developing Jewel lenses to be used as simple microscopes, before -in 1825- commissioning William Tulley to make him a triple achromatic lens of 0.33 inch focus, which heralds the true beginning of the achromatic period. Goring's non-achromatic object glass for low powers is mentioned as late as 1837 in Sir David Brewster's Treatise on the Microscope, with the addition: Mr Pritchard remarks, that when the focal length of the lens A (front lens) is not less than half an inch, this combination has been employed with considerable advantage, both as regards distinctness and aperture.

REDUCING CHROMATIC ABERRATION
Early in the development of the telescope and microscope, the problem of chromatic aberration took center stage. As early as 1733, Chester M. Hall discovered how to dramatically reduce chromatic aberration in the telescope objective by combining glass of two different refractive indices: crown glass and flint glass. This discovery was first used by the firm of Dollond to manufacture achromatic telescopes. However, making the tiny lens elements for microscopes using this principle was technically difficult at that time, given the limited tools available.

The Frenchman Dellebarre, who also worked in Holland for some time, attempted to get around this problem by combining four eyepiece lenses of different refractive index and colouring with conventional chromatic objectives, and whereby the eyepiece components could be screwed together in different combinations. The resulting image was essentially no better than other compound microscopes at the time (see the image of Dellebarre microscope No 35 in the Golub collection with exploded view of eye piece).First attempts at constructing achromatic microscope objectives were made by various workers in Europe, and these did produce better resolution, but only up to magnifications of up to 200x. Examples exist of objectives combining flint and crown glass, one made by Francois Beeldsnijder (1791)combining two biconvex lenses of crown glass with a biconcave flint glass lens in between, another by Jan and Harmanus van Deijl (1807)consisting of a planoconcave flint lens and biconvex crown glass lens, and a similar construction by Marzoli (1811).Most of these suffered from a number of defects, including poorly polished surfaces, spherical aberration, and loss of light due to reflection between the lens elements, which in these early designs were not cemented. Meanwhile, other workers chose to construct reflecting microscopes, which used concave mirrors, which - by nature- are achromatic, but these were difficult to make, adjust and maintain.

The Frenchman Selligue came up with a way of increasing magnification by using combinations of cemented achromatic doublets which were placed in series, and these were first constructed by Vincent and Charles Chevalier in Paris. In their original version, these had their convex (crown glass) surface facing the object, and it was found this resulted in too much spherical aberration, which was rectified by having the flat (flint) surface facing the object instead. These so-called "French button" combinations were initially made with between 2 and 4 achromatic doublets of the same focal length, and provided more than one magnification by using fewer or or more buttons screwed together, which inevitably increased spherical aberration. As explained below, correction of spherical aberration requires the placement of lens elements at a specific distance from one another, so that their aplanatic foci coincide, but at this stage no-one had as yet been aware of this necessity, and much was left to trial and error, so that some combinations were better than others.

Once Lister's work became more widely known, manufacturers such as Chevalier, Nachet, and Plossl, continued making button objectives, but made an attempt to combine aplanatic foci by using achromatic doublet combinations of different focal length, whereby the front lens had the shortest- and back lens the longest focus. These objectives could still be used with e.g. the front element removed to obtain a lower magnification, but otherwise were not meant to be taken apart, so that some makers started numbering their combinations, to ensure that their lens elements would be used in unison. The image on the left shows such a range of button objectives by Nachet from the mid 1850's.

By now the more prominent English microscope makers such as Andrew Ross and Hugh Powell had long abandoned this button type of construction for their objectives, but on the continent their production continued for a much longer period. There were also English opticians who continued to source the cheaper "French buttons" to make up their objectives, but managed to disguise them by incorporating these in an objective of English appearance as shown to the left, or, as shown to the right, simply by adding an outer cylindrical cover. The objective to the right was sold by Andrew Pritchard. Clicking on the image to left will reveal the hidden components of those two objectives.

Even after the introduction of the RMS standard objective thread in 1859, cheaper objectives continued to be sold to the English public, made up out of French buttons. In practice, their Numerical Aperture seldom exceeded 0.60, but in one of the author's collections is a 1/16 inch objective with a measured N.A. of 0.71.

REDUCING SPHERICAL ABERRATION
Spherical aberration occurs, when light rays which pass through the periphery (thinner part) don't focus in the same point as those which pass through the center (thicker part) of a lens, this, regardless of the colour of the light, so achromatic combinations also suffer from this problem. In a single achromatic combination consisting of a crown- and flint glass lens bonded together, spherical aberration can be reduced by stopping the aperture down, thereby excluding the peripheral rays from distorting the image, but this reduces resolution. Worse still, when more lenses are combined, they magnify each other's spherical aberration, so further stopping down would be required. From around 1825 it was realized, that a more scientific approach was required to measure the performance of microscope objectives, in order to be able to test the correction of spherical aberration in different lens combinations. The credit for coming up with the idea of test objects, so that the resolution of different objectives could be compared, belongs to Dr C.R. Goring. Examples of test objects (from The Micrographic Dictionary), are shown in the illustration to the left and include various objects with regular degrees of separation such as hairs, butterfly wing scales, and diatoms.

As noted earlier, the spacing of achromatic lens elements in objectives was first achieved by trial and error, but only in a gross qualitative way. With the use of Goring's test objects however, the results were much more quantifiable. The Italian optician Amici succeeded in this trial and error method to construct some of the best higher magnification objectives of the time, reaching numerical apertures of about 0.70. However, the scientific solution to this optical problem was provided by J.J. Lister in the late 1820's and reported publicly in his hallmark paper published in 1830 in The Philosophical Transactions (of The Royal Society). Lister shows, by using mathematical ray drawings, the path of light rays through each lens element, and concludes, that for each lens, there are two so-called aplanatic foci (f and f')whereby light rays emerging from these points are found to be free of spherical aberration. As such, for a lens combination to be aplanatic, i.e. free from spherical aberration, it is necessary to ensure, that the aplanatic foci of each lens coincide.If lens elements are spaced closer, or more distant, the combination will be respectively over- or under-corrected. The result is, that stopping down is largely not required, and the lens elements used can be employed at close to their full aperture.

Andrew Ross was the first major microscope maker to create objectives using Lister's formula. The progress he made can be illustrated by the observation that the angular aperture of his objectives reached 55 degrees by 1834 and 74 degrees in a 1/8 inch objective by 1842. The other major microscope makers soon followed suit.

ANGULAR APERTURE

The term Angular Aperture, shown diagramatically to the left as angle AOA, was first introduced by C.R. Goring, MD, in his paper: ‘On Achromatic Microscopes, with a description of certain Objects for their Defining and Penetrating Power”. Quarterly Journal of Science, 1827, pp.410-434. In the preceding years, he had been working with Tulley’s first achromatic objectives, and comparing them with those made by Chevalier, when he found, that the latter performed more poorly in terms of revealing fine detail on test objects e.g. butterfly scales, than those by Tulley. The explanation was soon found, when he realized, that the apertures were much further stopped down in the French objectives, which in turn, revealed more fine detail when their apertures were enlarged. Thus, he came to realize, that the functional diameter of the system, combined with the focal distance, i.e. the Angular Aperture, strongly correlated with the resolution.

This subject was expanded on in Goring’s collaboration with Andrew Pritchard in “The Natural History of the Animalcules” (1832), where -for the first time- they describe the method of measuring Angular Aperture of an objective using a candle and sector board (of the type reproduced by Jurriaan de Groot.). It then took a few more decades, before Abbe developed his theory of diffraction, and the refractive index of the medium was factored in, thus introducing the term ‘Numerical Aperture’. But even before the work of Abbe was published, various workers had discovered how to increase resolution by using immersion objectives.

SMITH'S QUARTERS:

Starting in 1839, James Smith formalized his independent business, making his own microscopes and achromatic aplanatic objectives. It was common for him, in his early years, to make microscopes with an extra front for higher magnification. These were sometimes referred to as Smith's Quarters. Later J.B. Dancer followed suit and also supplied this type of objective. The number 22 on this objective refers, unusually, to the serial number of the microscope to which this objective belongs.

THE CORRECTION COLLAR
EVOLUTION OF THE OBJECTIVE - THE CORRECTION COLLAR

Andrew Ross was one of the first to make achromatic aplanatic objectives. So good was the performance of these new objectives, that it was soon realized, that the use of a coverslip (then called a cover glass), added significant spherical aberration. This required a change in the spacing of the elements to account for this. To accomplish this, the front and back elements need to move closer to each other for thicker coverslips, and further away from each other for thinner ones (or none). In 1838 Andrew Ross was the first to make objectives where the spacing of the front and rear elements could be adjusted. In these first correctible objectives, an example of which is shown to the left, the front element was housed in a tube that was able to slide over a tube of smalller diameter containing the rear elements with small knobs provided to clamp the outer tube in any position once the desired correction was found.

Soon, this crude method of adjusting the spacing was replaced by a screw mechanism where turning the outer sleeve changed the position of the front element. The one difficulty with this arrangement is that this increased the total length of the objective, and hence increased the risk of damaging the front element by contact with the slide during the adjustment.

This Ross objective with correction collar has a separate, attached pointer to register the reading of correction on the collar's engraved indication marks. This is a rare form, as this was soon simply done with an engraved arrow on the collar and the numbers engraved on the fixed part of the objective.

In 1855,Wenham came up with a correction collar design in which the back elements were moved, so the front element retained the same position above the slide, greatly reducing the risk of front element damage. This type of correction collar, illustrated on a 19th century Ross objective as shown on the left, is still provided today on high power dry objectives and some water immersion objectives. In both types of coverslip adjustment, refocussing remains inevitable after the correction is made. The Ross objective shown here features a cone-shaped front; this was intended to allow more room for vertical illumination above an opaque specimen. This type of of front was popularised by Robert Tolles, and was also used by other makers such as Swift and Watson.

The use of correction collars has now become a "lost art" in modern microscopy. The few high N.A. dry- and water immersion objectives which still carry this fixture are marked with coverslip thickness markings, 0.17 mm being the norm, and usually ranging from 0.12 - 0.22 mm*. In the 19th century however, coverslip thickness tended to vary very widely even in the same batch, so frequent corrections were necessary, especially when examining diatom slides displaying fine markings with objectives of high N.A. As a result, users often recorded the numerical setting of the correction collar calibration mark on each slide when examined with an objective of a certain focal length,in order to reproduce the best resolution, as on English objectives these markings did not correlate with any pre-determined cover glass thickness. In order to obtain the best correction, and hence resolution for each object, there were a number of different methods which could be employed. The most basic of these was to align the linear markings for Uncovered and Covered shown on the objective, which is rather inprecise. Many users often employed the trial and error method, whereby with one hand on the correction collar, and the other on the fine adjustment control, the best setting was found which showed the best definition of a given object. Another method, advocated by Andrew Ross, and endorsed by Hugh Powell (in Description of the newly constructed achromatic microscope, 1842), was:

To place it at the point for viewing objects uncovered, which will be known by observing that the circular line under the word uncovered corresponds with the fixed line; or:, the more ready way is, to adjust it down as far as it will allow, as we always make them to stop at the corrected point. Bring the object into focus by adjustment of the body, then adjust the object-glass till the upper surface of the glass which covers the object is in focus; this can very readily be done while the person is observing, by taking between finger and thumb the milling on the object-glass, and turning it to the left; then bring the object again into focus by the body, and the adjustment is perfect

While this method works well for objectives with a longer working distance such as a 1/4 inch, in practice, those with a shorter focal length can not be focused on the object while in the uncovered position,as coverglass thickness prevents this, and the opposite procedure is required: set the correction on fully covered, focus on the top of the coverslip, and then turn the correction collar down, i.e. a bit more towards the uncovered position in order to bring the object into focus. One finds that with objectives of a very short focal length and working distance, the object can still not be brought into focus even with the correction collar in the maximum covered position. These objectives were made to be used with extra thin cover glasses, such as those which Powell & Lealand commissioned from Messrs Chance, which reportedly were only 0.05 mm thick.

*Standard coverslips today, although closer to their stated thickness, are not exactly their stated thickness however; they vary within a range, and so when doing precise high power work, just setting a correction collar on the expected number may not suffice and some adjustment may be neccessary. Some more exactly standardized coverslips are available, but these are extremely expensive. A chart of modern coverslip thicknesses shows the usual ranges for standard coverslips today.

Over time, more scientific methods were developed to define the point of optimum correction for these objectives. Abbe introduced the test plate(left) for checking spherical correction. It consisted of a set of six small cover slips of different measured thicknesses mounted side by side on a microscope slide and having their under surfaces silvered and ruled to produce parallel bright and dark bars. The microscope was brought into focus on the bars under one cover in a narrow cone of axial light, and the illumination was then made oblique by racking the closed iris diaphragm to the margin of the condenser aperture (a feature on Zeiss microscopes). If the focal level of the image remained constant, this demonstrated that the full aperture of the objective was working uniformly; if the marginal focus differed from the axial focus, the full lens aperture was not being constructively used towards creating the image. The thicker covers introduced an error in one direction, the thinner ones in the opposite, and by adjusting the correction collar (or the tube length), an objective could be matched to any of them. The later version of the test plate used a single long coverslip, tapering in thickness from end to end, with the thickness engraved on the slides at points along the length(right).

For most purposes, the Abbe test plate has been superceded by the simpler and more sensitive star test. This compares the out-of-focus appearances of a small bright point in the image above and below the level at which the Airy disc has its minimum diameter.These tiny bright spots can be created by examining a cover slip, the under surface of which has been silvered, leaving small perforations. For the testing of immersion objectives, the top of the slide itself is silvered. The star test depends on the fact that if the objective is free from spherical aberration, the distribution of light in the discs seen above and below the point of focus ought to be identical.If the central focus of the objective is shorter than its marginal focus - i.e. if it is over-corrected- the light will form a bright spot below the true focal plane, and a ring above it, owing to the uneven distribution of light rays. The reverse effect occurs with an undercorrected lens. The correction collar is then used to get an as symmetrical effect as possible, i.e. the same appearance above- and below the focal point. As an alternative, adjustments can be made to the length of the draw tube (see diagram). In principle, this test can also be performed when focusing on a small dark speck in the slide to be examined against a bright background, and examining the out of focus images above and below the focal plane, but this requires a bit more practice.

IMMERSION AND THE BALSAM ANGLE
One of the things that was realized in the 19th century, was that spherical aberration worsens with every change in the medium that the light is transmitted through. Hence, the glass-air-glass interface between the slide coverslip and the objective front lens increased aberration and this aberration was proportionate to the difference in refractive index between the two mediums (glass and air). It was soon realized that placing a fluid of higher refractive index than air between the glass of the coverslip and the objective would reduce this effect. Since the refractive index of air is 1.0, and glass is close to 1.5, any liquid with a higher refractive index than air will improve the situation, and the closer to the refractive index of the glass in question, the greater the improvement.

Water, with a refractive index of 1.33, was the first obvious liquid to try and water immersion objectives were made early on. In fact, Hooke tried water immersion briefly as early as 1678, Although immersion of objectives was tried by Hooke and suggested by Sir David Brewster, immersion objectives using water were first made by Amici in 1843, though with the purpose of reducing aberration, not improving numerical aperture. In 1859 Hartnack made his first water immersion objective, and this led to its popularity; he was the first to add a correction collar to a water immersion objective. This kind of water immersion objective, a 1/8th inch by John Browning is shown to the left. Note the setting or Covered and for Wet, the latter indicating water immersion, and the angular aperture of 135^o, which corresponds to a numerical aperture of 1.23 when used as an immersion lens in water, or 0.924 in air-its not clear which this was referring to.

In 1867 Ernst Gundlach may have been the first to make glycerin(refractive index about 1.47) immersion objectives. Robert Tolles was the first to use a fluid with the same refractive index as glass, softened Canada Balsam, for oil immersion in 1871. One of the problems in assessing the angle of aperture arose when immersion techniques began to be used frequently and that is that angular aperture is not a linear scale and also is not the same for different refractive indices of the medium that is between the objective and the slide; the angular aperture of a dry objective cannot be compared to an immersion objective. For this reason, at least in America, makers often referred to a balsam angle or B.A. for the angular aperture of an immersion objective in balsam and this was sometimes inscribed on the objectives of the time, and for some time afterwards, as seen on the Herbert Spencer Objective shown to the left. By 1870, Zeiss, with the help of Abbe, was making all their objectives by calculation; in 1873, the year that Abbe published his scientific work on the resolution of objectives relating to numerical aperture, Tolles made his famous 1/10 inch homogenous immersion objective with a N.A. of 1.25. But Balsam was hard to work with and hardened with time and so was not ideal. Tolles made a glycerine immersion objective that same year. In 1877 Abbe, after testing hundreds of liquids, discovered that Cedarwood oil has a refractive index(1.516) close to glass, and all his oil immersion lenses were then designed to work with this; within a few years Cedarwood Oil replaced softened Canada balsam for all oil immersion objectives.

Powell & Lealand made their first oil immersion objective, a 1/12, in 1879. Their first 1/50 oil immersion objective, with the serial number of 1, is shown to the left, and was made just one year later in 1880 with the published n.a. of 1.38. By this time it had become known that a 1/50" focal length objective would not improve resolution over a 1/12 with the same n.a., but apparently the demand for such objectives did not cease.

Although cedarwood oil was used until the 1940's it has drawwbacks, including absorbtion of UV and yellow light, hardening with time, and damaging the cement of objectives if left on the lens too long. It also turns yellow with age; it is considered nontoxic however. In the 1940's synthetic immersion oils were developed by Cargille which did not harden nor turn colors. Although these oils seemed to be a big improvement, one drawback was that they contained carcinogenic PCBs (polychlorinated biphenyls) until 1971. Commercial immersion oil of today no longer contains these compounds. A little known fact is that the refractive index of immersion oils varies with temperature. Most modern oils are designed to be used at a 23^oC (about 73.4^oF).

NUMERICAL APERTURE
In the 1870s Ernst Abbe became the first to be able to combine all previous knowledge with his own mathematical abilities to produce completely predictable configurations to dramatically reduce chromatic aberration and spherical aberration and maximize resolution through the use of homogenous immersion where the immersion solution is not only used between the objective and the slide, but also thje condenser and the slide. In addition he was able to produce a formula that allowed the comparison of the resolving power of all objectives regardless of whether they were dry or immersion lenses. This term he called the numerical aperture (n.a.) and for the first time, different types of objectives could, by their n.a., be directly compared. He also designed the famous Abbe apertometer to directly measure the n.a. of any objective without the need to do any calculation. The term n.a. is still used today to give the user a way to compare the resolving power of different objectives. Not only could one express the n.a. of both oil and dry objectives, but the scale is linear so an n.a. of 0.8 means twice the resolution of an n.a. of 0.4. n.a. is calculated by the formula:

n.a.= n sin α

where n=refractive index of the medium between the subject and the objective, and α is half the angle of aperture.

ONGOING USE OF IMMERSION MEDIA:
As noted above, water, with a refractive index of 1.33, was the first regularly-used immersion media. Glycerin offered a slight improvement with a refractive index of 1.47, while cedarwood oil is 1.516, and modern immersion oils match the n.a. of glass nearly exactly without the drawbacks of cedarwood oil. Interestingly though, glycerin was used in the mid-20th century as an immersion media for flourescence microscopy, because it does not flouresce like many oils do. Water immersion continues to be used even today when the specimen itself is live and in water.

Objectives that could be used with more than one media were made starting in the 19th century. Among the most interesting of these were objectives made by Robert Tolles who, for an additional fee and upon request, supplied two fronts for his better objectives, one dry and one for immersion. The cans for these objectives had an extra compartment on the bottom to house the extra front. Some objectives could be used wet or dry, with the correction collar so indicating the range of corrections for each.

In the 20th century, Zeiss even made objectives with correction collars indicating ranges of correction for 3 different media-oil, glycerin, and water.

VARIABLE MAGNIFICATION OBJECTIVES
On occasion makers would produce variable magnification objectives. These lenses have adjustment collars resembling correction collars, but these collars are actually used to vary magnification. Examples shown here were made by Crouch and Zeiss.

OBJECTIVES TO INCREASE CONTRAST-DARK GROUND MICROSCOPY

Although staining of specimens was developed early in the developement of microscopy, stains are both inconvenient and may interfere with the cellular functions of living specimens. In order to visualize details of specimens that were studied in solution, often the solutions they occur in naturally, and have a refractive index close to the solution (mainly water), other techniques were developed to visualize details in these specimens. Perhaps the earliest methods were Dark Ground (also called dark field) illumination, and Oblique Illumination. Neither of these require special objectives at low power, but at high power the field of view of the objecive must be stopped down to match the illumination. High power dark ground illumination was especially important for rapidly detecting spirochetes(right) in the skin lesions of patients with Primary Syphilis. This required a special condenser and either a funnel stop for an ordinary objective, or later,an iris diaphragm built in to the objective. These tiny iris diaphragms are difficult to make and were not common in objectives until the middle of the 20th century.

OBJECTIVES TO INCREASE CONTRAST-PHASE CONTRAST:
The 20th century saw a renaissance in new ways to increase contrast. The earliest of these new techniques requiring special objectives was Phase Contrast. So great an achievment was this, that Hans Zernicke, its inventor, was awarded the Nobel Prize in Physics. Zernicke invented the technique in 1932, and it was commercially introduced by Kohler and Loos in 1941. Phase contrast is still used today, in fact one of us (BJS), uses a phase contrast microscope to examine urine specimens almost every day. Phase contrast objectives have a phase ring and require matching phase rings in the condenser. A Wild phase contrast condenser and a 40X Wild Fluotar Phase Contrast objective are shown here to the left. Typically phase contrast objectives are made in 10X, 20X, 40X and 100X(oil) magnifications. An image of a Wild M-20 microscope outifitted for phase contrast is on this site. Wild Fluotar objectives are among the best ever made.

OBJECTIVES TO INCREASE CONTRAST-DIFFERENTIAL INTERFERENCE CONTRAST (DIC AND NIC):

Another technique for increasing contrast in specimens like this is Differential Interference Contrast or DIC, and also Nomarski IC or NIC. DIC was invented prior to 1947 by Francis Hughes Smith in England, who submitted his British patent in 1847, and his US patent in August of 1948. It utilized a polarized light microscope with Wollaston prisms added to the front focal plane of the condenser, and another at the rear focal plane of each objective. Each objective had its own specially oriented prism. These objectives are labeled specifically Interference Contrast or, as in the German Leitz objectives seen here, Interf. Kontrast. There is a manual available for the use of the Leitz apparatus on this site.

Soon after Smith's description, Georges Nomarski patented his improvement in France in 1952; his American patent was filed in May of 1953 and approved in February of 1960. Nomarski IC allowed the prisms, of unique thin construction, to be located away from the aperture conjugate planes in the condenser, and allowed the use of a single prism in the optical tube above the objective to be used with different strain-free objectives. These prisms are referred to as Nomarski prisms. By the 1970's NIC became a standard way to increase contrast, and although cheaper than Smith's DIC, it was still expensive, and because of its cost, it never completely replaced phase contrast. Smith DIC or NIC have some advantages over phase contrast, notably the elimination of the halo around objects seen with phase contrast, but are much more expensive to produce. Since DIC depends on polarized light, objectives for DIC must be made with lenses that do not change polarization of light. These are often labeled as POL or S (for strain-free) if used with NIC; as noted above Smith DIC objectives, (still strain-free) are labeled with the words Interference Contrast because they have built-in prisms. In addition to strain-free objectives, NIC requires the use of a (NIC) condenser, AND also a NIC intermediate tube containing an adjustable prism, placed between the binocular head and the optical tube. Shown to the left is an olympus combined phase contrast and NIC condenser, along with a typical Olympus 20X S-plan PL objective, that can be used for both phase contrast and DIC work. Note the condenser has an N.A. of 1.4, suitable for high power homogenous oil immersion work. Unlike phase contrast, DIC images have a color imparted to them and also appear three dimensional. The color can be varied by the user to optimize contrast for the particular subject. An Olympus Vanox microscope equipped for NIC as well as phase contrast, is on this site.

OBJECTIVES TO INCREASE CONTRAST-HOFFMAN MODULATION CONTRAST (HMC):

Still later, about 1975, Hoffman Modulation contrast was developed. This technique also imparts a three-dimensional appearance to the subject, but does not impart color. Its advantages include the fact that it is less expensive to produce than NIC, and it can be used with plastic containers which cannot be used with NIC or Smith DIC, due to their effects on polarized light. It requires special dedicated objectives and condensers. A modulator is placed the rear focal plane of the objectives. Such objectives are labeled with either HMC or the words Hoffman Modulation Contrast spelled out, or simply Modulation.

OBJECTIVES TO INCREASE CONTRAST-DISPERSION STAINING
In 1948 S. C. Crossmon, N. B. Dodge, and co-authors R. C. Emmons and R. N. Gates all wrote papers on the use of dispersion effects through the microscope to characterize particles. This technique imparts color to colorless transparent specimens immersed in high refractive index liquids by dispersion of part of the spectrum of polarized light by using a stop placed at the back focal plane of the objective and matching the stop to the opening of the aperture diaphragm By the 1950's dispersion staining with polarized light, became popular to detect asbestos and this has been the standard method for many years. A special objective is used for this and is usually labeled as a Dispersion Staining Objective, sometimes abbreviated DSO. Using this technique, crysotile asbestos with appear blue or magenta depending on the orientation of fibers. These objectives have facility for changing from a central stop to a central small aperture.

VARIABLE MAGNIFICATION OBJECTIVES:

Occasionally, objectives with variable magnification were produced. Such objectives have adjustment which indicates the different magnifications. An example by Zeiss is shown to the left and one by Crouch to the right.

VERTICAL ILLUMINATION:
When viewing opaque objects through a microscope, illumination is required from above. For low power work, this is a relatively simple matter, but for higher power work, as the working distance becomes shorter and shorter, an alternative method is required. For small opaque objects, a Lieberkuhn reflector, reflecting light from below the stage back down onto the object is feasible. But for larger opaque objects this is not possible.

As time went on, imaging of larger opaque objects at high power, especially metals, became important. Initially this was accomplished with illumination from the the side, but a direct view from the vertical plane was needed, and became critical at higher powers where side illumination was not satisfactory. The vertical illuminator, placed above the objective, and using the objective both as a condenser and objective was used. When examining uncovered objects like metal, they were used with objectives designed to be used without a coverglass at all, either by setting a correction collar to the uncovered position, or using objectives designed solely for uncovered use; such objectives became known as metallographic objectives.

In the 19th century,Robert Tolles was among the first to construct objectives with built-in prism-type vertical illuminators. An example of this specialized objective is shown to the left and a schematic diagram of it to the right (both courtesy of Allan Wissner).

What were called episcopic or Epi- illumination systems were first developed by Zeiss in the early 20th century. In this type of illumination a directed and focused beam of light surrounds the objective and is arranged such that the optimal plane of illumination conincides with the focusing plane of each objective. These objectives direct highly oblique light on the subject to provide dark ground illumination to opaque objects. These objectives provide a concentric path around the outide of the objective optics that receive the image. An example of a Wild Epi objective is shown here to the left. Note it is much wider than a standard objective because of the accessory light path outside the optics that receive the image.

Early on, metallographic objectives were not labeled directly as such, but could be recognized by their shorter barrel length. Such objectives with various kinds of vertical illuminators were used well into the 20th century and eventually had the letter M inscribed on their barrel, as in the M-plan 40X objective by olympus seen to the left. The previously mentioned technique of DIC was also adapted to vertical illumination. A DIC vertical illumination Olympus Vanox microsccope is in this collection and will be featured on this site in the future.

INSCRIPTIONS ON OBJECTIVES:
The reader may have noticed that various immersion objectives may be labeled slightly differently. Furthermore, some details may only be on the can and some only on the objective. To help make sense out of these I provide the following guidance.

INSCRIPTION	USUAL MEANING
immersion	water immersion
wet or dry	dry or water immersion
oil	oil immersion
gl gli or gly	glycerin (=glycerol) immersion
w	water
HI	homogenous immersion (=oil immersion, including condenser)
xxxo	angular aperture in degrees
B.A.	balsam angle (=angular aperture in canada balsam)
n.a. or N.A	numerical aperture
Fl	Flourite
PL	Plan (flat field)
NPL	normal field of view, flat(planar)
Holos	Holoscopic (Watson)
Tubus or T.L.	Mechanical Tube Length
Achr or Achro	achromatic
Para	Parascopic(Watson)
Apo	Apochromatic
planapo	flat field apochromatic
THE FOLLOWING EXAMPLES ARE FOUND MAINLY OBJECTIVES DATING FROM THE 2ND HALF OF THE 20TH CENTURY:
INSCRIPTION	USUAL MEANING
S or P or Po or Pol or SF	strain free, low birefringence for polarized light work)
Fluotar	Flourite
Ph, PHACO, PC	phase contrast objective
DL,DM	phase contrast: dark low, dark medium
PLL, PL	phase contrast: positive low low, positive low
NL,NM,NH	phase contrast: negative low, negative medium, negative high contrast (higher contrast regions appear lighter)
BD	brightfield or darkground
EPI	episcopic (vertical) illumination
F	focal length
WD	working distance
DIC or IC	differential interference contrast
NIC	Nomarski Differential Interference Contrast
Interf. Kontrast	interference contrast-usuall Smith type (German Notation)
M	metallographic (without coverslip)
Neo	Dark Ground vertical illumination(Zeiss) (not to be confused with Neofluar which are strain free high transmission objectives)
U	Can be for ultraviolet light microscopy or alternatively for use with a universal stage
UV	Ultraviolet transmitting objective
DI, MI or TI	Interferometry, noncontact, mutliple bear (Tolanski)
HMC	Hoffman Modulation Contrast

In many cases there are numbers without an abbreviation occur such as 160/0.17 indicating intended mechanical tube length and thickness of coverslip, both in mm. As indicated above a number with the degree notation, like 110^o indicates the angle of aperture.